My lab recently purchased a monoclonal antibody of the peptide Galanin from Sigma-Aldrich. According to the user's manual, the band should be found at about 38kD. The resultant Western blot picture was clean and didn't have much problems, until one day we realised that the GST tag that was added to the original protein shouldn't have an effect on the molecular weight of endogenous Galanin, which only has 30 amino acids (galanin precursor is 123 aa). So I want to know, should we be worried by this fact? My boss is going bonkers already, but I'm still hanging on to the belief that there's a scientific explanation to this band position. The link to the above-mentioned antibody is listed below.
http://www.sigmaaldrich.com/catalog/product/sigma/wh0051083m1?lang=en®ion=CA
Hi there,
if you detect the fusion with GST the band is indeed around 38KD but if your intention is to detect your protein alone you should get a much lower band (around 12kD for the precursor and around3kD for the mature protein)...
The galanin polypeptide that was conjugated to GST was the full-length 124-residue preproprotein, which would have a molecular weight of around 14,000. The GST tag (26,000) is irrelevant, since it should not be present in your source material, and the mAb should not recognize it. The Western blot that Sigma shows is detecting the fusion protein construct they used as the antigen, which has no relevance to your use of the mAb. What you should see is either the 14 kDa full-length galanin precursor polypeptide or the processed 3200 molecular weight neuropeptide. If you see a higher molecular weight band, it is most likely due to a cross reaction of the antibody with some other protein.
The Western blot was performed by separating the (GST-GAL) on an SDS-PAGE. This is a very very poor control. The blot does not even proof that the monoclonal antibody is raised against the galanin precursor. Even if this is the case it would be important to know whether you can expect to detect the 30 amino acid protein. In the best case you may be able to detect the precursor and the peptide. However, you should not detect the 38kD band.
My recommendation: Contact the technical service and ask for additional information, e.g. references where the antibody has been used.
Sigma antibody looks fine. It picks up a band of 38kD, which reflects the MW of GST-124aa recombinant protein. If matured Galanin has only 30 aa as you said, for you western, you should only see a western band around mw of 30 aa position. If in your western, you see a band around the position of 38kD, there must be other reasons. Non-specific recognition, Galanin modification etc could be the reason. Or even the antibody may pick up GST like protein in your sample as sigma does not indicate the 38 band is due to the recognition of matured galanin (30aa) but not due to GST, or the left peptide outside of the matured galanin 30 aa in the 124 aa.
As far as I understand, the immunogen from Sigma was preprogalanin with 123 aa (~13K) connected with GST tag (26K) resulting in their WB band of ~39K. In the Figure to their picture they indicated that that was a cell lysate from the cell line producing the original immunogen - preprogalanin + GST tag (39K). The mature form of galanin has only 30 aa (~3K). Is it possible that under conditions you run your gel for WB the mature form of endogenous galanin was moving with the front line on the gel and was not detected. I would recommend to run a gel with higher acrylamide concentration. You might also see different galanin-related peptides (with different MW) as a result of alternative splicing of the original gene (if they are recognized by your monoclonal Ab). For more information please see the link below:
https://en.wikipedia.org/wiki/Galanin
"The galanin family of protein consists of four proteins, of which GAL was the first to be identified. The second was galanin message-associated protein (GMAP), a 59- or 60-amino acid peptide also formed from the cleavage of preprogalanin.[10] The other two peptides, galanin-like peptide (GALP) and alarin, were identified relatively recently and are both encoded for in the same gene, the preproGALP gene. GALP and alarin are produced by different post-translational splicing of this gene."
The information is all there on Sigma's webpage (in cursive):
The galanin precursor has 123 amino acids (= molecular mass of 13,302):
MARGSALLLASLLLAAALSASAGLWSPAKEKRGWTLNSAGYLLGPHAVGNHRSFSDKNGLTSKRELRPEDDMKPGSFDRSIPENNIMRTIIEFLSFLHLKEAGALDRLLDLPAAASSEDIERS
The immunogen is the galanin precursor plus GST (= molecular mass of ~39,000):
Immunogen
GAL (AAH30241, a.a. 1-124) full-length recombinant protein with GST tag. MW of the GST tag alone is 26 kDa.
and the immonogen was also used in the Western blot to validate the antibody:
Immunoblotting
Monoclonal Anti-GAL, Cat. No. WH0051083M1 used at the antibody concentration: 1 μg/mL. Specific band of ~39.27 kDa using immunogen protein lysate.
Therefore, it is quite right that their Western shows a ~39 kD band. They did not use galanin or galanin precursor to validate their antibody!
I think that people are saying that, if the antibody recognises GST and not GAL then the result would be the same on Sigma's Western. They should be showing a blot from a cell line or other tissue which expresses the protein to prove specificity. As Uwe says, blotting a synthesised immunogen is a very poor control. Just because the antibody recognises the antigen doesn't prove specificity. It could also recognise any number of related (or indeed unrelated) proteins. I think you do have a problem - it is good to remember that many antibody distributors do not test the antibodies themselves in the techniques they recommend and, increasingly, antibodies are very poorly characterised before selling. Only those used for diagnostic purposes and therefore rigorously tested can be assumed to be specific. Try sourcing your antibodies through a trusted research institution who do test their antibodies - they will at least be able to point you in the direction of decent antibodies. This is not a problem unique to Sigma. There is a recent paper in MAbs by the European Antibody Network which may be of interest.
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